The focus of this proposal concerns the molecular pathogenesis of meningitis due to Cryptococcus neoformans. C. neoformans is an encapsulated yeast that is a major pathogen in the immunocompromised population. The principal clinical manifestation of infection with C. neoformans is meningitis, and this is uniformly fatal if untreated. Persons immunosuppressed as a result of infection with the Human Immunodeficiency Virus (HIV), an underlying malignancy or treatment with corticosteroids are at particularly high risk for developing cryptococcal meningitis. The population of these patients is ever-increasing, yet current treatment regimens are associated with poor clinical success rates and high toxicity. New antifungal agents with activity against C. neoformans are needed. In this project, basic molecular research on this yeast will be used to identify genes important for pathogenesis. Identification of these genes and their products can the lead to the development of new treatment strategies for cryptococcal meningitis. Two main strategies will be used to identify genes important in pathogenesis. The first involves complementation of avirulent mutant with plasmids containing genomic DNA from the virulent wild type parent strain. Transformants will be selected in vivo for restoration of virulence, and the plasmids will be recovered for analysis. The second strategy will select for promotor sequences that are preferentially transcribed in vivo rather than in vitro. Previous data from this laboratory have shown that the ADE2 mutants are avirulent in the rabbit cryptococcal meningitis model, but virulence can be restored by complementation with a functional ADE2 gene. Constructs will be made by ligating small fragments of genomic DNA to a truncated ADE2 gene. These constructs will then be used to transform ADE2 mutants, and colonies that are pink (and therefore not expressing the ADE2 gene at high levels in vitro) will be used to infect rabbits. Virulent transformants in which the promotor has been turned on by in vivo conditions will be selected by survival in the host, and the promotor sequences will be recovered. These recovered promotor sequences will then be used to probe a genomic library to identify the entire genes. The genes found using both strategies will then be sequenced and used in gene disruption studies to test for effects on pathogenicity. The disrupted genes that result in loss of virulence will be considered as candidates for further study.